Method and System for Treating Gastro-Esophageal Reflux Disease (GERD)

ABSTRACT

A system and method for reducing the likelihood of GERD includes the modification of an individual&#39;s gut microbes in a manner that reduces, if not eliminates, the symptoms of GERD. Certain embodiments employ Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated system (CRISPR-Cas) or Clustered Regularly Interspaced Short Palindromic Repeats from  Prevotella  and  Francisella  1 (CRISPR/Cpf1) system to render  H. Pylori  more susceptible to certain drugs, including antibiotics, thus addressing the antibiotic resistance otherwise experienced by treating  H. Pylori  with antibiotics.

RELATED APPLICATIONS

This application is a non-provisional of U.S. Provisional PatentApplication Ser. No. 62/274,550, filed on Jan. 4, 2016.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 14/752,192 filed Jun. 26, 2015, which is acontinuation-in-part application of U.S. patent application Ser. No.14/225,503 filed Mar. 26, 2014, (now issued U.S. Pat. No. 9,445,936,issued Sep. 20, 2016), which is a continuation of U.S. patentapplication Ser. No. 13/367,052, filed Feb. 6, 2012 (now issued U.S.Pat. No. 8,701,671, issuing on Apr. 22, 2014), which claims priority ofU.S. Provisional Patent Application Ser. No. 61/439,652, filed on Feb.4, 2011 and U.S. Provisional Patent Application Ser. No. 61/556,023,filed on Nov. 4, 2011.

This application also is a continuation-in-part application of U.S.patent application Ser. No. 15/270,034, filed Sep. 20, 2016, which is acontinuation-in-part application of U.S. patent application Ser. No.14/954,074, filed on Nov. 30, 2015 (now issued U.S. Pat. No. 9,457,077,issuing on Oct. 4, 2016), which is a continuation-in-part application ofU.S. patent application Ser. No. 14/574,517, filed on Dec. 18, 2014 (nowissued U.S. Pat. No. 9,408,880, issuing on Aug. 9, 2016), which claimspriority of U.S. Provisional Patent Application Serial Nos. 62/072,476,filed on Oct. 30, 2014; 62/053,926, filed on Sep. 23, 2014; 62/014,855,filed on Jun. 20, 2014; and 61/919,297, filed on Dec. 20, 2013.

This application is also a continuation-in-part application of U.S.patent application Ser. No. 15/228,454, filed Aug. 4, 2016.

This application claims priority of U.S. Provisional Patent ApplicationSer. No. 62/275,341 filed Jan. 6, 2016.

This application is also a continuation-in-part application of U.S.patent application Ser. No. 14/611,458, filed Feb. 2, 2015, which is acontinuation-in-part application of U.S. patent application Ser. No.14/502,097, filed Sep. 30, 2014 (now issued U.S. Pat. No. 9,010,340,issuing on Apr. 21, 2015), which is a continuation of U.S. patentapplication Ser. No. 14/307,651, filed on Jun. 18, 2014 (now issued U.S.Pat. No. 8,936,030, issuing Jan. 20, 2015), which is acontinuation-in-part application of U.S. patent application Ser. No.14/079,054, filed Nov. 13, 2013 (now issued U.S. Pat. No. 8,757,173,issuing on Jun. 24, 2014), which is a continuation of U.S. patentapplication Ser. No. 13/425,913, filed Mar. 21, 2012 (now issued U.S.Pat. No. 8,584,685, issuing on Nov. 19, 2013), and claims priority ofU.S. Provisional Patent Application Ser. No. 61/467,767, filed Mar. 25,2011.

The entire disclosure of the prior applications are considered to bepart of the disclosure of the accompanying application and are herebyincorporated by reference.

FIELD OF THE INVENTION

A system and method for reducing the likelihood of GERD includes themodification of an individual's gut microbes in a manner that reduces,if not eliminates, the symptoms of GERD. Certain embodiments employCRISPR-Cas or Cpf1 systems to render H. Pylori more susceptible tocertain drugs, including antibiotics, thus addressing the antibioticresistance otherwise experienced by treating H. Pylori with antibiotics.

BACKGROUND OF THE INVENTION

Gastro-Esophageal Reflux Disease (GERD) is a chronic heartburn/stomachacidity affliction characterized by a series of symptoms includingheartburn, chronic indigestion (dyspepsia), pyrosis, abdominal pain andswelling, epigastric pain, nausea, regurgitation, premature feeling ofsatiety, acid reflux coughing, vomiting and gas indigestion. GERD is adisease that mainly consists of abnormal refluxing of gastric contentsinto the esophagus through the lower esophageal sphincter, the lowestportion of the esophagus where it joins the stomach, which prevents thereverse passage of gastric content. The pH of the gastric content isacid, which is why the feeling of heartburn is produced and when itreaches the throat or the mouth, the taste is acidic and bitter, asymptom known as pyrosis. GERD is presently the most prevalent diseasein the upper digestive system, and it is very prevalent in the Westernworld. It is estimated that at least 30 million patients suffer one ofthe aforementioned forms of GERD in the US each year, with the numberrising significantly every year. The greatest incidence occurs in adultsover forty. H. pylori is a gram-negative, slowly growing bacteria strainadapted to microaerophilic environments and its presence in the stomachis directly associated with various gastric diseases. 75% of gastriccancers are associated H. pylori infection.

Since Helicobacter pylori (initially called campylobacter) wasdiscovered in 1980, it has been considered as a major cause in thepathogenesis of gastric ulcer, mucosa-associated lymphoid tissue (MALT)lymphomas, and gastric cancer. Eventually antibiotics were designed toeradicate this bacterium, which not only prevent peptic ulcer recurrencebut also decrease the chances of developing gastric cancer. Around 50%of the world's human population is infected with Helicobacter pylori, aclass I carcinogen. Eradication of H. pylori results in healing ofgastric ulcer and may also reduce the incidence of gastric carcinoma.While various drug regimens have been used for the eradication of H.pylori such as triple or quadruple drug therapy, there has been analarming increase in the resistance to antibiotics.

Helicobacter pylori can cause a chronic low-level inflammation of yourstomach lining and is a major factor in the symptoms of acid reflux.Colonization of the human stomach by Helicobacter pylori and its role incausing gastric cancer is one of the richest examples of a complexrelationship among human cells, microbes, and their environment. It isalso a puzzle of enormous medical importance given the incidence andlethality of gastric cancer worldwide. There is a long felt but unsolvedneed to address GERD in a fashion that avoids the over administration ofantibiotics, leading to increased antibiotic resistance.

Proton pump inhibitors (PPI's) work by blocking the production of acidin the stomach. In response, the body reacts by overcompensating, oftenrevving up production of acid-making cells. Thus, one problem involvedin attempting the cessation of PPI's is that individuals experienceexcess growth of acid producing cells in the stomach, so that when theyunblock production of acid by stopping PPI doses, they have more of theacid-making cells, thus creating more acid production than they hadpreviously experienced. The widespread treatment of GERD has also failedto reduce the incidence of esophageal cancers, with esophagealadenocarcinomas, which are associated with GERD, increasing by 350percent since 1970. Thus, when people take PPI's, they do not cure theproblem of reflux—but rather, they have just controlled the symptoms.

Stomach acid is needed to break down food and absorb nutrients, as wellas for proper functioning of the gallbladder and pancreas. Taking PPI'schanges the ecology of the gut and actually allows overgrowth of somethings that normally would be kept under control. Long-term of use ofPPI's may interfere with these processes and suppression of stomachacid, which kills bacteria and other microbes, may make people moresusceptible to infections, like C. difficile, which can make one moresusceptible to a dangerous infection marked by diarrhea, abdominal painand even death.

C. difficile kills an estimated 14,000 to 30,000 people a year in theUnited States. Evidence of the link between C. difficile and PPIs wassufficiently strong that the Federal Drug Administration has issuedalerts that PPI's may be causing a 65 percent increase in C.difficile-related diarrhea, that they may potentially lower magnesiumlevels—a condition that can cause muscle spasms, irregular heartbeat andseizures—and that they increase the risk of hip, wrist and spinefractures.

Clostridium difficile is a Gram-positive, strictly anaerobic,spore-forming bacterium found in mammalian intestinal tracts. C.difficile is one of the key public health problems in industrializedcountries and one of the major nosocomial enteropathogens. C.difficile-associated diarrhea is currently the most frequently occurringnosocomial diarrhea worldwide. Two major risk factors for contracting C.difficile infections are the age of the individual and exposure toantibiotics. Antibiotic therapy causes alterations in the colonicmicroflora, allowing the development of C. difficile from preexisting oracquired spores. The pathogen synthesizes two major toxins, TcdA andTcdB, which glucosylate host GTPases, resulting in alterations in theenterocyte cytoskeleton. This induces intestinal cell lysis andinflammation, resulting in diarrhea, pseudomembranous colitis, and evendeath.

The long-term use of Prilosec and other proton-pump-inhibiting drugs israising concerns as to the safety and wisdom of such long-term drug useas it invariably has consequences with respect to human health. PPI'sare the third highest-selling class of drugs in the United States, withmore than 100 million prescriptions and over $15 billion in sales. Someconsider the long term use of proton pump inhibitors like Prevacid andPrilosec and the H2 blocker agents like Tagament, Pepcid, and Zantac tobe dangerous. Such drugs significantly reduce the amount of acidavailable to digest food and the reduction of acid in the stomach alsodiminishes a primary defense mechanism for food borne infections, thusleading to an increased risk of risk of food poisoning.

Long-term use of PPI's can make it difficult to absorb some nutrients.Rebound acid hypersecretion, defined as an increase in gastric acidsecretion above pre-treatment levels following antisecretory therapy,has been observed within two weeks after withdrawal of treatment and canlead to acid-related symptoms and possibly PPI dependency. Individualshave attempted to wean themselves off of these drugs gradually or elsethey experience a severe rebound of symptoms, often resulting in theproblem being worse than it was before they started taking themedication. Emerging evidence suggests that PPIs are not as benign asonce thought, with newer data implicating a potential association ofPPIs with an increased risk of respiratory tract infections,gastrointestinal infections, bone fractures, hypomagnesemia, and theoccurrence of rebound hyperacidity after discontinuation of PPI therapy.

In recent years, there have been numerous warnings about long-term useand high doses of PPI's being associated with an increased risk of bonefractures and infection with a bacterium called Clostridium difficilethat can be especially dangerous to elderly patients. Studies have shownlong-term PPI use may reduce the absorption of important nutrients,vitamins and minerals, including magnesium, calcium and vitamin B12, andmight reduce the effectiveness of other medications, with the F.D.A.warning that taking Prilosec together with the anticlotting agentclopidogrel (Plavix) can weaken the protective effect (of clopidogrel)for heart patients. PPIH is the consequence of intestinal Mg²⁺malabsorption. An underappreciated aspect of PPIH is frequent secondaryelectrolyte disturbances such as hypocalcemia and hypokalemia. Theclinical significance of reduced calcium (Ca²⁺) levels is emphasized byseveral dozens of studies showing increased risk of bone fractures afterchronic PPI use. Omeprazole inhibits passive paracellular Mg²⁺ fluxes,predominantly present in the small intestine and omeprazole directlyinterferes with important transcellular Mg²⁺ transport mechanisms of thecolon.

The human gut harbors one of the most complex and abundant ecosystemscolonized by more than 100 trillion microorganisms. Firmicutes andBacteroidetes represent more than 90% of the relative abundance of thegut microbiome. The phylum Bacteroidetes is composed of Gram-negative,non-spore forming anaerobic bacteria that tolerate the presence ofoxygen but cannot use it for growth. Actinobacteria (e.g.,Bifidobacterium) are Gram-positive, multiple branching rods, non-motile,non-spore-forming, and anaerobic bacteria. The most abundant genera fromthe Bacteroidetes phylum are Bacteroides and Prevotella species. As thegut is an anaerobic environment, aerobic pathogenic species cannotinvade and colonize it, but anaerobic and facultative pathogenic speciescan nevertheless invade it, causing diseases. High diversity defineshealthy human gut microbiomes, whereas reduction in diversity isassociated with dysbiosis—referring to an imbalance in the microbiomestructure that results from an abnormal ratio of commensal andpathogenic bacterial species.

There is therefore a long felt but unsolved need for effective methodsand compositions to reduce the likelihood of GERD and in treating,mitigating and preventing GERD, especially in concert with thewidespread use of PPI's.

SUMMARY OF THE INVENTION

Humans possess an inflammatory response—the triggering of theoverproduction of hydrochloric acid—as the stomach's primary response tobacterial colonization. Inflammation of the stomach lining coincideswith production of peptides called cytokines, which stimulate productionof a hormone called gastrin. Gastrin triggers parietal cells in thestomach lining to produce more hydrochloric acid, which kills off mostinvading microbes. Notably, H. pylori is the only bacterial organism inthe stomach that cannot be killed by hydrochloric acid. If you inhibitgastric acid production, you interfere with the stomach's naturaldefense mechanism. An abnormally low level of acidity in the stomach isa factor in various disease states. Since reduced gastric acidity doesappear to make the mammalian stomach more vulnerable to bacterialinvasion and gastritis, however, physicians are advised to re-evaluatethe long-term use of proton-pump-inhibiting drugs in their patients.

One aspect of the several embodiments of the present invention isdirected to the modification of microbes in a manner that reduces, ifnot eliminates, the symptoms of GERD. Helicobacter pylori is one ofseveral pathogens that persist within the host despite a robust immuneresponse. H. pylori elicits a proinflammatory response from hostepithelia, resulting in the recruitment of immune cells which manifestsas gastritis. Certain embodiments employ CRISPR-Cas or Cpf1 systems torender H. pylori more susceptible to certain drugs, includingantibiotics, thus addressing the resistance otherwise experienced bytreating H. pylori with antibiotics.

While not bound by theory, it is believed that H. pylori survivesantimicrobials, including calprotectin (CP), which employs nutrientsequestration, through alteration of its outer membrane. Thus, oneembodiment of the present invention relates to the interference with andmodification of the normal mechanism of H. pylori resistance toantibiotics by affecting the ability of H. pylori to form biofilms,including the retention and maintenance of lipid A production, which isnormally interfered with by H. pylori when contacted by CP. Inparticular embodiments, CRISPR-Cas systems are employed to undermine theability of H. pylori to form biofilms. One such strategy is to addadditional genetic components in H. pylori cultures that include lipid Aexpression and the purposeful inclusion of such a culture of H. pyloriin a person's stomach so as to establish a competitively beneficialculture of such modified H. pylori in the stomach. Thus, once suchbacteria are the predominant bacteria, as compared to non-modified H.pylori, then the application of CP (or other suitable antibiotics) canbe used to eliminate or vastly reduce the number of H. pylori bacteriain the person's stomach. Having the individual provided with further H.pylori cultures that possess such modified characteristics is furthercontemplated as a way in which to preclude the reestablishment of a wildtype H. pylori culture from persisting in the person's stomach. By sucha strategy, the person is able to substantially eradicate H. Pylorispecies that are resistant to antibiotics. Other ways to decrease theformation of H. pylori biofilms include increasing cell surfacehydrophobicity. Another way is to enhance the function of the Lpx lipidA biosynthetic enzymes (e.g. LpxF, LpxL, and LpxR enzymes) to ensurethat their functions are not perturbed. Thus, to combat H. pyloriresistance to cationic antimicrobial peptides, one target is to affectthe formation of biofilms and to reduce the ability of H. pylori tomodify endotoxins. This can be achieved in various ways, but preferablyby employment of CRISPR-Cas systems to interfere with genes involved inthe formation of biofilms by H. pylori.

Certain embodiments are directed to the modification of resident H.pylori populations in vivo in a person's stomach so as to beneficiallydisrupt the colonization of the gastric glands by H. pylori. Otheraspects involve the modification of dietary components and essentialmicronutrients in concert with the gastrointestinal microbiota to affecta beneficial modification of H. pylori activity so as to maintain it asa commensal bacteria and to prevent its activity as a pathogen, thusprecluding its carcinogenic potential.

One aspect of the present invention is directed to therapeuticinterventions in the microbiome directed against molecular entities,such as essential and antibiotic resistance genes to quorum sensingsystems components used to control microbial networking behaviors,including the chemical communication and production of virulencefactors. Various embodiments are focused on dietary interventions andmicrobial modification genetic tools to modify and/or eliminatepathogenic microorganisms and to control dysbiosis. Various embodimentsof the present invention are also directed towards the modification ofthe human-microbiota ecosystem to promote health and to combat disease,including the modification and/or elimination of certain bacteria livingin the human body. The determination of human microbiota and theanalyses of the presence or absence of specific microbial species inaccordance with particular diseases provides one of skill in the artwith the ability to identify particular biomarkers and to target thesame to treat GERD.

While phage therapy could potentially have beneficial impact on humanmicrobiomes, host specificity greatly limits the types of bacteria thatcan be employed and the selection of a specific phage to use as atherapeutic agent requires in-depth knowledge of the pathogen causing agiven disease. In the absence of such knowledge, some have suggested theuse of a cocktail of different species of phages to broaden the range ofaction, but such a cocktail could have undesired negative effects on themicrobial community. Thus, in preferred embodiments of the presentinvention, CRISPR systems are employed to effect desired microbialmodifications. The relative simplicity of the mechanism of action andthe peculiarities of Cas9 make the CRISPR/Cas9 system an ideal tool fora vast assortment of procedures, particularly for genomic editing, andin various embodiments of the present invention, the editing ofbacterial strains is employed to interfere with the development of GERDand to otherwise treat GERD.

In various embodiments of the present invention, various targets forintervention using CRISPR-Cas systems include the modification ofbacteria resident in the human gut that are distinct from humans invarious respects. For example, most bacteria synthesize thiamine denovo, whereas humans depend on dietary uptake. Methionine is notsynthesized de novo in humans and must be supplied by diet. In contrast,most bacteria need to synthesize methionine to survive. There are amyriad of other orthologous gene groups conserved in both human andhuman commensal gut microflora that are not suitable targets for drugdevelopment. The majority of unique targets found in microbes' genomesare genes responsible for the metabolism of carbohydrates, amino acids,xenobiotics, methanogenesis, and the biosynthesis of vitamins andisoprenoids, and in particular for the purposes of various embodimentsof the present invention, focus is directed to those genes that arenon-homologous to those encompassed in human genome. A number ofmicrobial genes and products, including bacteriocins, lysins, holins,restriction/modification endonuclease systems, and other virulencefactors contribute to resistance to antibiotics. Thus, an alternative tokilling or inhibiting growth of pathogenic bacteria is targeting thesekey regulatory systems. Other aspects of the present invention aredirected to targeted changes in microbiota by the rational use ofprebiotics and probiotics to abolish metabolic alterations associatedwith various maladies, including GERD, obesity, cancer, etc.

In particular embodiments, compounds such as halogenated furanonesproduced by many microbial species, mostly belonging to theproteobacteria, are employed to interfere with AHL and AI-2 QS pathwaysin Gram-negative and Gram-positive bacteria. It is believed that byinterrupting normal systems of bacterial inter and intra quorum sensing,one may effectively modify bacterial cell-cell communication in a mannerthat prevents colonization by pathogenic bacteria, and in particular,can be employed to interfere with biofilm formation by H. pylori andthus, treat GERD.

High doses and frequent use of antibiotics can disrupt and destabilizethe normal bowel microbiota, predisposing patients to developClostridium difficile infections. Up to 35% of these patients develop achronic recurrent pattern of disease. Fecal bacteriotherapy is thetransplantation of liquid suspension of stool from a donor (usually afamily member) and has been used successfully in severe cases ofrecurrent C. difficile relapse. Many problems exist with this therapysince it can increase the risks of transmitting other pathogens. Oneparticular focus of the present invention is to employ transplantedmicrobiota to treat metabolic disorders in humans but that limit therisks involved in conventional fecal transplants. For example, via theuse of modified bacteria (e.g. using CRISPR-Cas systems) one is able tomore effectively employ antibiotics to target particular regions of thebody, to target particular bacteria, etc. in a manner that avoids thecomplications experienced in the typical use of antibiotics, which causecomplications from C. difficile growth.

In particular embodiments, probiotic microorganisms that possessresistance to low gastric pH and have the capacity to reach theintestines alive, are used to exert beneficial effects on the humanbody, preferably lactic acid-producing bacteria of the Lactobacillus andBifidobacterium genera. Such microorganisms are preferably thosemodified using CRISPR-Cas to provide a population that can be moreeasily controlled and manipulated to maintain particular levels in anindividual's microbiome. Specifically, some embodiments of the presentinvention include regulating the balance of intestinal microbiota byphysically blocking the adhesion of pathogenic species onto epithelialcells, such blocking action directly mediated by means of increases inthe production of a mucosal barrier by goblet epithelial cells and/or byregulating epithelial permeability by enhancing the formation oftight-junctions between cells. Use of CRISPR-Cas to modify or deletevirulence factors of particular bacteria, such as adhesion abilitiesthereof, is employed in this fashion.

In various embodiments of the present invention, CRISPR/Cas9 is used toselectively deplete a given bacterial community of a particular harmfulstrain or species, or of particular virulence factors possessed byparticular strains of bacteria. Thus, in certain embodiments, theidentification of a harmful pathogen is performed, and CRISPR/Cas9 isthen used to selectively deplete or modify that particular bacterialspecies from an individual's gut microbiota. The use of antibiotics isbelieved to increase the ability of bacteria to acquire drugresistance-encoding plasmids. Thus, the CRISPR/Cas9 system may be usedto introduce specific mutations into essential, antibiotic resistanceand virulence genes, as well as to directly modulate the expression ofparticular genes. For example, one can employ a Cas protein that lacksnuclease activity but retains a binding capacity so as to repressbacterial transcription by binding to promoter regions to effect theblocking of transcriptional initiation and/or elongation. CRISPR-Cas orCpf1 systems may also be used to fuse regulatory domains in order toswitch on/off the expression of specific genes. Thus, the presentinvention includes the engineering of commensal bacteria with improvedproperties using a CRISPR/Cas system to prevent and treat diseases. Oneof skill in the art will appreciate the steps required to affect thedesired levels of target specificity and delivering efficiency.

Still other embodiments employ the modification of various beneficialbacteria so that they express certain compounds and substances, notablythose substances found to be effective as an anti-H. pylori agent, suchas those isolated from garlic and ginseng. Allicine, the main activemolecule present in Garlic extract, is used to effect immune modulation.The use of dialkyl-thiosulfinate and/or propy thiosulfonate can beemployed to improve disease resistance of a pathogen, with thesecompounds generated from the natural degradation of propiin, a moleculepresent in most Allium species, and more specifically onion, shallots orchives. Still other sulfur compounds can may be employed to inhibit H.pylori colonization. Using CRISPR-Cas, one is able to modify residentbacteria to express the active ingredient in garlic found to be aneffective killer of H. pylori. Such expressed compounds include thosedescribed above.

Similarly, another aspect of the present invention is to control H.pylori populations in an individual's stomach by a diet includingkimchi. Other aspects are directed to the expression of kimchi genes byone or more bacterial species that reside in a human stomach. In amanner similar to the expression of certain genes derived from garlic,one is therefore able to control the population of H. pylori in aperson's stomach. The use of CRISPR-Cas to insert genes into particularbacteria so as to facilitate the control of H. pylori is one aspect ofcertain embodiments, including the insertion of genes having the activeagent contained in Korean kimchi and garlic. The incidence of gastriccancer is about 20 per 100,000 population (in Korea) and about 50 per100,000 population (in Japan, where far less kimchi is eaten),demonstrating that kimchi is effective as a cancer preventative agent.Similarly, the expression of garlic related genes by one or morebacterial species that reside in a human stomach is another embodimentof the present invention. The use of garlic (as well as kimchi) toaddress GERD is considered to be a teaching away from the prior art, asmany have identified garlic and onions as causing heartburn. Garlic(allium sativum), like onions, shallots and leeks, among others, belongsto the alliaceae family, and all contain organosulfur products. Garlicin particular contains allicin, an organosulfur compound that isproduced when garlic is broken or crushed, through the action of theallinase enzyme on alliin. Allicin is a potent phytocide, with markedantibiotic and antifungal properties. The release of allicin producesother sulfur derivatives, such as ajoene, allyl sulfides, diallylsulfides, allyl methyl disulfide, allyl methyl trisulfide, s-allylcysteine and diallyl trisulfide. Allicin pronouncedly inhibits thesecretion of various cytokines (IL-1b, IL-8, IP-10 and MIG) fromepithelial cells, suppressing the expression of interleukin 8 (1-8) andinterleukin 1 b (IL-1b) mRN and is therefore considered to be effectivein attenuating intestinal inflammation. It is believed that low doses ofgarlic oil suppresses NOS (inducible Notric Oxide Synthase) activity,ulceration and apoptosis of the intestinal mucosa. At high doses,however, garlic oil has shown a toxic effect, which is why it is deducedthat garlic is beneficial in moderate doses, but can be toxic in highdoses. Garlic extracts and garlic oil have also been found to bepowerfully anti-microbial against other GI bacteria such as Escherichiacoli, Shigella sp, Salmonella sp, and Proteus mirabilis.

One aspect of the present invention is directed to the use of probioticsto modulate the human microbiota and promote health and preventantibiotic side effects. L. species are acid-resistant and commensal andtheir concentrations in the normal human stomach vary between 0 and 10³mL⁻¹. They can survive in the stomach for periods of up to 2 h. Invarious embodiments, fructo-oligosaccharides (FOS) andtrans-galacto-oligosaccharides (TOS), such as inulin, are used toselectively stimulate growth and activity of health-promoting bacteria.In this regard, dietary inulin fibers are used to stimulate Mg²⁺ andCa²⁺ absorption and are a potent stimulant of mineral absorption,especially achieved by oligofructose-enriched inulin. Certain strains ofgut bacteria have a preference for inulin fibers. Thus, one aspect ofthe present invention is to selectively advance the population of suchbacteria in a person's gut. It is known that N-butryic acid increasesCa²⁺ and Mg²⁺ absorption. Thus, certain embodiments of the presentinvention are directed to the provision of bacteria designed to producen-butryic acid to treat PPI-induced Ca²⁺ disturbances. It is believedthat dietary inulin stimulates intestinal Mg²⁺ absorption, and thus,other embodiments of the present method include the provision of dietaryinulin in addition to the provision of the various bacteria strains asdescribed herein. One aspect of the present invention is thereforedirected to the impact of PPIs on Ca²⁺ homeostasis and provides atreatment for PPI-induced mineral disturbances. Dietary oligofructoseenriched inulin fibers are believed to prevent omeprazole-inducedreduction of Ca²⁺ absorption and lead to improved intestinal Mg²⁺absorption, thus preventing PPI-induced mineral deficits in individuals.

In various embodiments, the present invention is directed to the use ofdietary inulin to counteract reduced intestinal Ca²⁺ absorption upon PPItreatment. One aspect of the present invention is directed to the localluminal acidification of the colon to enhance intestinal Mg²⁺ absorptionand by so doing, preventing PPIH. Other embodiments are directed to theuse of the fructan fiber inulin to reduce intestinal pH, such ingestedinulin fibers being fermented in the large intestine by bifidogenic gutbacteria, resulting in short-chain fatty acids (SCFA), which in turnacidify the colon. Thus, one aspect of various embodiments is directedto the stimulating action of SOFA on intestinal Mg²⁺ absorption byreducing the luminal pH. Certain aspects are directed to the enhancementof intestinal Mg²⁺ and Ca²⁺ absorption in order to counteractomeprazole-induced defects in mineral uptake. Proton-pumpinhibitor-induced hypomagnesemia (PPIH) is the most recognized sideeffect of proton-pump inhibitors (PPIs). Additionally, PPIH isassociated with hypocalcemia and hypokalemia. It is hypothesized thatPPIs reduce epithelial proton secretion and thereby increase the pH inthe colon, which may explain the reduced absorption of and Mg²⁺ andCa²⁺. Fermentation of dietary oligofructose-enriched inulin fibers bythe microflora leads to acidification of the intestinal lumen and bythis enhances mineral uptake. One aspect of the present invention istherefore directed to the improvement of mineral absorption byapplication of dietary inulin to counteract PPIH.

In various embodiments of the present invention, candidate probioticstrains are isolated from fecal samples, especially after enrichmentwith a prebiotic application. As described in other applicationsincorporated herein, the use of particular fecal samples from healthyAmish individuals is employed to combat GERD. Moreover, one strategy forenhancing the establishment of probiotic bacteria in the humanintestinal tract is via the parallel administration of a prebiotic. Invivo selection (IVS) may be employed to isolate candidate probioticstrains from fecal samples following enrichment with a prebiotic. Forexample, isolated bifidobacteria from human subjects who consumedincreasing doses of galactooligosaccharides (GOS) revealed an 8-foldenrichment in Bifidobacterium adolescentis strain IVS-1. It is believedthat such selected strains are able to outcompete residentBifidobacterium populations. One aspect of the present invention,especially employing one or more of the modified bacteria as describedherein, is to substantially enhance the establishment andcompetitiveness of one or more putative probiotic strains in anindividual's gastrointestinal tract to combat GERD.

One aspect of the present invention is directed to the provision ofpharmaceuticals based on an individual's own microbiome. Thus, incertain embodiments, isolation of particular bacteria from anindividual's stool is employed and CRISPR-Cas and/or Cpf1 systems arethen used to modify such bacteria in various beneficial ways, asdescribed herein. The reintroduction of such modified bacteria into theperson's gut (e.g. via fecal transplantation) provides a way toselectively and competitively compete with other undesired bacteria inthe person's gut. Preferably, the resident populations of gut microbesare reduced substantially before the reintroduction of the modifiedbacteria, thus providing a better chance and opportunity for theestablishment of a population of preferred bacteria, as modified via theCRISPR-Cas systems, as described herein.

The growth of microbiota communities is under control of distinctsubfamilies of host genes encoding antimicrobial peptides (AMPs). Whenbacteria colonize a given human habitat, the expression of AMPs,including α and β defensins and cathelicidins, is upregulated in orderto limit the spreading of bacteria. The equilibrium between the immunesystem and immunoregulatory functions of bacteria appears to be adelicate balance in which the loss of a specific species can lead to anoverreaction or suppression of the innate immune system. The maintenanceof a stable, fermentative gut microbiota requires diets rich in wholeplant foods particularly high in dietary fibers and polyphenols.Individuals colonized by bacteria of the genera Faecalibacterium,Bifidobacterium, Lactobacillus, Coprococcus, and Methanobrevibacter havesignificantly less of a tendency to develop obesity-related diseaseslike type-2-diabetes and ischemic cardiovascular disorders. Thesespecies are characterized by high production of lactate, propionate andbutyrate as well as higher hydrogen production rates, which are known toinhibit biofilm formation and activity of pathogens. Thus, in variousembodiments of the present invention, these bacterial species areselected and administered to an individual in preferred ratios thatreflect those of healthy individuals so as to attain the general balanceof bacterial populations in a person's gut. Moreover, preferablybacteria are selected that are effective in inhibiting biofilm formationand in particular, those that demonstrate a high production of lactate,propionate, butyrate and hydrogen. CRISPR-Cas and/or Cpf1 may beemployed to provide such characteristics to the selected bacterialspecies in this regard.

CDT (Clostridium difficile transferase) is a binary, actinADP-ribosylating toxin frequently associated with hypervirulent strainsof the human enteric pathogen C. difficile, the most serious cause ofantibiotic-associated diarrhea and pseudomembranous colitis. CDT leadsto the collapse of the actin cytoskeleton and, eventually, to celldeath. The lipolysis-stimulated lipoprotein receptor (LSR) is the hostcell receptor for CDT. By applying the CRISPR-Cas technology tointerfere with the binding component of CDT, preferably by impactingamino acids 757 to 866 of CDT, one is able to interfere with the bindingof CDT to the LSR. Thus, interfering with the interaction between CDTand its receptor LSR, is one way to provide an anti-toxin strategy forpreventing cell entry of the toxin. Use of the active expression ofCRISPR arrays in C. difficile strains is therefore one way in which tocounter Clostridium difficile nosocomial infections associated withantibiotic therapies. One aspect of certain embodiments is directed tomodifying the site where the bacterium Clostridium difficile's binarytoxin binds to intestinal cells' LSR (lipolysis-stimulated lipoproteinreceptor) protein and triggers a mechanism that results in the invasionof the host cells by the bacteria. Clostridium difficile produces thebinary, actin ADP-ribosylating toxin CDT (Clostridium difficiletransferase). While CDT can lead to death of the host cells throughcollapse of the actin cytoskeleton, low doses of CDT result in theformation of microtubule-based protrusions on the cell surface thatincrease the adherence and colonization of C. difficile. Thus, oneaspect of certain embodiments relates to the interference with theadherence characteristics of this bacteria by reducing the amount of CDTproduced by bacterial cells, thus providing a population of C. difficilethat do not pose the problems of wild type strains. One aspect ofcertain embodiments involves blocking certain areas in the toxin and thereceptor in order to prevent the Clostridium difficile transferase toxinfrom entering the host cell.

One will appreciate that this Summary of the Invention is not intendedto be all encompassing and that the scope of the invention nor itsvarious embodiments, let alone the most important ones, are necessarilyencompassed by the above description. One of skill in the art willappreciate that the entire disclosure, as well as the incorporatedreferences, pictures, etc. will provide a basis for the scope of thepresent invention as it may be claimed now and in future applications.

BRIEF SUMMARY OF THE DRAWINGS

FIG. 1 illustrates a normal stomach as compared to a GERD stomach,showing that the sphincter is closed in a normal stomach, whereas thesphincter is open in a GERD stomach, thus permitting reflux to occur.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE PRESENT INVENTION

In various embodiments of the present invention, bacterial species to beexposed to a person's gut microbiome, include those specificallymodified by employing the CRISPR-Cas and CRISPR-Cpf1 systems to renderthe virulence factors of various bacteria ineffective. CRISPR (ClusteredRegularly Interspaced Short Palindromic Repeats) is a prokaryoticadaptive defense system that provides resistance against alien repliconssuch as viruses and plasmids. CRISPRs evolved in bacteria as an adaptiveimmune system to defend against viral attack. Upon exposure to a virus,short segments of viral DNA are integrated into the CRISPR locus. RNA istranscribed from a portion of the CRISPR locus that includes the viralsequence. That RNA, which contains sequence complimentary to the viralgenome, mediates targeting of a Cas9 protein to a target sequence in theviral genome. The Cas9 protein cleaves and thereby silences the viraltarget. In preferred embodiments, rather than using CRISPR-Cas, oneemploys the CRISPR-associated endonuclease Cpf1. e.g. a CRISPR fromPrevotella and Francisella 1 (Cpf1) nuclease for CRISPR-based genomeediting. Prevotella sp. C561 SEQ ID NO: 184 250 67 357 425 78 357 425 78gi|345885718|ref|ZP_08837074.1; Prevotella timonensis CRIS 5C-B1 SEQ IDNO: 170 208 39 328 375 61 328 375 61 gi|282880052|ref|ZP_06288774.1 (andincorporating 20150252358 to Maeder by this reference).

CRISPR-Cpf1, a class II CRISPR effector that is distinct from Cas9, is asingle RNA-guided endonuclease that uses T-rich PAMs and generatesstaggered DNA double stranded breaks instead of blunt ends. Its smallerprotein size and single RNA guide requirement makes CRISPR applicationssimpler and with more precise control.

In some preferred embodiments, the present invention provides methodsfor the use of CRISPR loci to determine the potential virulence of aphage against a cell and the use of CRISPR-cas to modulate the geneticsequence of a phage for increased virulence level.

Certain embodiments of the present invention are directed to theinterplay between periodontitis and GERD, with the belief that the oralmicrobiome and the microbiome of an individual's stomach and gut arerelated and affect the overall health of the individual, including butnot limited to the occurrence of GERD. Thus, various embodiments of thepresent invention are directed to a method for protecting a person fromperiodontitis and related illnesses associated therewith involvinggenerating a bacterial oral microbiome culture comprising T. denticolaand/or Prevotella bacteria modified by employing CRISPR-Cas or Cpf1comprising the steps of: (a) exposing a parent bacterial strain of oneof c T. denticola and/or Prevotella comprising at least a portion of aCRISPR locus to at least one bacteriophage to produce a mixture ofbacteria comprising at least one bacteriophage resistant variant straincomprising a modified CRISPR locus comprising at least one additionalspacer in said modified CRISPR locus; (b) exposing a parent bacterialstrain comprising at least a portion of a CRISPR locus, different to theparent bacterial strain of step (a), to a different at least onebacteriophage to that of step (a) to produce a mixture of bacteriacomprising at least another bacteriophage resistant variant straincomprising a modified CRISPR locus comprising at least one additionalspacer in said modified CRISPR locus; (c) selecting said bacteriophageresistant variant strains from said mixtures of bacteria; (d) selectingsaid bacteriophage resistant variant strains comprising an additionalspacer in said modified CRISPR locus from said bacteriophage resistantstrains selected in step (c); and (e) isolating said bacteriophageresistant variant strains, wherein said strains comprise an additionalspacer in said modified CRISPR locus.

Spacers in a CRISPR cassette confer immunity against viruses andplasmids containing regions complementary to the spacers and hence, theyretain a footprint of interactions between prokaryotes and their virusesin individual strains and ecosystems. The human gut is a rich habitatpopulated by numerous microorganisms, each having a CRISPR system. Tocomply with written description and enablement requirements,incorporated herein by the following references are the following patentpublications: 20140349405 to Sontheimer; 20140377278 to Elinav;20140045744 to Gordon; 20130259834 to Klaenhammer; 20130157876 to Lynch;20120276143 to O'Mahony; 20150064138 to Lu; 20090205083 to Gupta et al.;20150132263 to Liu; and 20140068797 to Doudna; 20140255351 to Berstad etal.; 20150086581 to Li; PCT/US2014/036849 and WO 2013026000 to BRYAN.

The CRISPR-Cas system may also be employed to render certain bacteriasensitized to certain antibiotics such that specific chemical agents canselectively choose those bacteria more susceptible to antibiotics, see,e.g. US Pat. Publication No. 2013/0315869 to Qimron, which isincorporated in its entirety by this reference.

The microbiome of an individual is disrupted by antibiotics and thus,the employment of CRISPR as a way to bypass common modes of multidrugresistance, while being selective for individual strains, is employed invarious embodiments of the present invention to attain the benefitsderived by the presence of particular bacteria.

Another aspect of certain embodiments includes making syntheticCRISPR-containing RNAs that target genes of interest and using them withCas enzymes. The specificity of CRISPR-Cas systems permits one to designmethods to target a single bacterial species so that only essentialgenes form that one species is targeted and cut up. CRISPR-Cas systemsare employed in various ways in the many embodiments of the presentinvention to retain the beneficial bacterial communities intact and tooffer protection against undesired bacterial pathogens.

CRISPR has a certain protein in it called Cas9 that acts like a scissoras it recognizes specific sequences of DNA and cuts it enabling one toperform genome-editing of a bacterial genome in a person's microbiome.There exists another CRISPR system, CRISPR-Cpf1 that is even morepreferred for use in microbial systems.

Cpf1 is important in bacterial immunity and is well adapted to slicetarget DNAs. Cpf1 prefers a “TTN” PAM motif that is located 5′ to itsprotospacer target—not 3′, as per Cas9, making it distinct in having aPAM that is not G-rich and is on the opposite side of the protospacer.Cpf1 binds a crRNA that carries the protospacer sequence forbase-pairing the target. Unlike Cas9, Cpf1 does not require a separatetracrRNA and is devoid of a tracrRNA gene at the Cpf1-CRISPR locus,which means that Cpf1 merely requires a cRNA that is about 43 baseslong—of which 24 nt is protospacer and 19 nt is the constitutive directrepeat sequence. In contrast, the single RNA that Cas9 needs is still˜100 nt long. Cpf1 is apparently directly responsible for cleaving the43-base cRNAs apart from the primary transcript.

With respect to the cleavage sites on the target DNA, the cut sites arestaggered by about 5 bases, thus creating “sticky overhangs” tofacilitate gene editing via NHEJ-mediated-ligation of DNA fragments withmatching ends. The cut sites are in the 3′ end of the protospacer,distal to the 5′ end where the PAM is. The cut positions usually followthe 18th base on the protospacer strand and the 23rd base on thecomplementary strand (the one that pairs to the crRNA). In Cpf1 there isa “seed” region close to the PAM in which single base substitutionscompletely prevent cleavage activity. Unlike the Cas9 CRISPR target, thecleavage sites and the seed region do not overlap. One advantage of thepresent invention, as compared to techniques that rely on CRISPR systemsto modify mammalian cells, is that the system and method of preferredembodiments are directed to bacterial systems—rather than eukaryoticsystems. It is believed that Cpf1 may be better than Cas9 for mediatinginsertions of DNA, namely because its guide RNA is only 43 bases long,making it feasible to purchase directly synthesized guide RNAs for Cpf1,with or without chemical modifications to enhance stability.

The CRISPR system may be employed in various embodiments to strengthenantibiotics or to kill the bacteria altogether. By removing thebacteria's genes that make them antibiotic-resistant, CRISPR can boostthe effectiveness of existing drugs. CRISPR can also be used to remove abacteria's genes that make them deadly and facilitate RNA-guidedsite-specific DNA cleavage. Analogous to the search function in modernword processors, Cas9 can be guided to specific locations within complexgenomes by a short RNA search string.

In various embodiments, the CRISPR-Cas systems is employed to controlthe composition of the gut flora, such as by circumventing commonlytransmitted modes of antibiotic resistance and distinguishing betweenbeneficial and pathogenic bacteria. For applications that require theremoval of more than one strain, multiple spacers that target shared orunique sequences may be encoded in a single CRISPR array and/or sucharrays may be combined with a complete set of cas genes to instigateremoval of strains lacking functional CRISPR-Cas systems. Because of thesequence specificity of targeting, CRISPR-Cas systems may be used todistinguish strains separated by only a few base pairs.

As described above, certain aspects of the present invention address thedifferences of C. difficile and other bacteria, especially with respectto the understanding that the CRISPR-Cas system in C. difficile isunique due to the presence of multiple active CRISPR arrays, which is incontrast to the presence of silent or barely expressed CRISPR loci insome other bacteria such as Streptococcus pyogenes and E. coli. Inrelation to the C. difficile infection cycle, stress conditions,including antibiotic treatments, induce prophages and lead to therelease of phage particles and infection of neighboring bacteria, thuscontributing to the CRISPR spreading within C. difficile populations.Together with dysbiosis, this can increase the rigor of this pathogen.Thus, employment of CRISPR-Cas to excise particular characteristics ofC. difficile to defeat, for example, the ability to release phageparticles, and to then provide such modified bacteria to a person's gut,thus maintaining the balance as referred to therein, is one way in whichto modify a person's gut microbiota in a beneficial manner without thelong term use of PPI's or antibiotics, thus avoiding the dangers andproblems associated therewith.

The specificity of targeting with CRISPR RNAs may be employed to readilydistinguish between highly similar strains in pure or mixed cultures.Thus, in certain embodiments, varying the collection of delivered CRISPRRNAs is employed to quantitatively control the relative number ofindividual strains within a mixed culture in a manner to circumventmultidrug resistance and to differentiate between pathogenic andbeneficial microorganisms.

In certain other aspects, particular embodiments of the presentinvention are directed to the use of CRISPR to excise certain priorinfectious virus DNA sequences that are considered responsible for theincreased obesity of individuals harboring the same. Reference is madeto Kovarik, U.S. Pat. No. 8,585,588, “Method and system for preventingvirus-related obesity and obesity related diseases,” incorporating thesame by this reference. After determining whether one has been infectedwith a particular virus, the viral DNA can then be excised viaCRISPR-Cas to remove the previously inserted DNA, thus effectivelyreducing if not eliminating the virus gene from the individual.Thereafter, to avoid being infected with such virus again, practice ofthe method as set forth in U.S. Pat. No. 8,585,588 will lessen, if notprevent, reacquisition of such virus.

Use of CRISPR-Cas provides a generalized and programmable strategy thatcan distinguish between closely related microorganisms and allows forfine control over the composition of a microbial population for use inthe present invention. Thus, the RNA directed immune systems in bacteriaand archaea (e.g. their CRISPR-Cas systems) is employed in variousembodiments of the present invention to selectively and quantitativelyremove and/or alter individual bacterial strains based on sequenceinformation to enable the fine tuning of exposure of desired antigens.Thus, such genome targeting using CRISPR-Cas systems allows one tospecifically remove and/or alter individual microbial species andstrains in desired ways.

In various embodiments, it is desirable to remove—using CRISPR-Cassystems—particular viable genes in pathogenic bacteria and/or otherpathogenic portions (e.g. plasmids, etc. of such bacteria)—while sparingother desired commensal bacteria, in order to provide exposure todesired immune developing proteins.

In various embodiments, one of skill in the art will appreciate thatremoval or alteration of particular strains of bacteria may be achievedusing both type I and type II CRISPR-Cas systems, given the distinctionbetween these systems being that type I systems cleave and degrade DNAthrough the action of a 3′-to-5′ exonuclease, whereas type II systemsonly cleave DNA. In still other embodiments, multiple guide RNAs canalso be used to target several genes at once. The use of effectorfusions may also expand the variety of genome engineering modalitiesachievable using Cas9. For example, a variety of proteins or RNAs may betethered to Cas9 or sgRNA to alter transcription states of specificgenomic loci, monitor chromatin states, or even rearrange thethree-dimensional organization of the genome.

There are ongoing ethical concerns arising with respect to the use ofCRISPR-Cas systems—especially as it relates to modification of the humangenome. Still others are concerned about the chain reactions possible ifundesired gene expression is directed to insects (mosquitoes, etc.) orinvasive species, etc.—such that as yet unknown problems may arise bythe removal or alterations of problems presently experienced. Inpreferred embodiments of the present invention, however, such issues aremuch less prevalent for various reasons. First, because preferredembodiments relate to the modification of microbes—rather than to thehuman genome—and especially only those microbes that show tropism forhumans, the unintended consequences of employing Crispr-Cas on organismsis lessened, if not eliminated. Moreover, use of CRISPR-Cas to alsoinsert genes that have controllable elements such that the cells arekilled by triggering the expression of such genes, is another way toreduce if not eliminate concerns about an unintended release of amodified organism. These types of controls are well known to those ofskill in the art and have been long employed, for example, by thoseinvolved in creating genetically engineered organisms, such as byinserting genes so that organisms become susceptible to variousconditions, such as temperature, antibiotic exposure, etc., such thatmicrobes that may somehow escape desired conditions will not be viable.

On aspect of many embodiments of the present invention include the useof specialized viruses to supply CRISPR/Cas to rid bacteria ofantibiotic-resistance plasmids and/or other virulence factors. Virulencefactors of Gram-negative anaerobes such as Prevotella include, forexample, fimbria, hemolysins, adhesions and hemagglutinins. Thesebacteria commonly produce immunoglobulin-degrading enzymes and someproduce tissue-degrading enzymes. Additionally, bacteria of the genusPrevotella are often resistant to antibiotics, such as tetracycline,erythromycin, and β-lactam antibiotics. In practice, aPrevotella-targeting lambda phage is created that encodes the CRISPRgenes plus spacers that target two conserved β lactamases, enzymes thatconfer resistance to β-lactam antibiotics. Once integrated into thePrevotella genome, the phage prevents the transfer of βlactamase-encoding plasmids and can also delete these plasmids fromindividual bacterial cells. These lambda phage-encoding bacteria thenbecome sensitive to treatment with antibiotics.

In other embodiments, the use of CRISPR-Cas systems is employed toincrease butyrate production of these bacteria. For example, F.prausnitzii, one of the most abundant species in the colon, is animportant producer of butyrate, a major product of carbohydratefermentation which is implicated in providing protection againstcolorectal cancer and ulcerative colitis.

Modifying the human genome, made possible by the CRIPSR technique, hasits own wonderful upsides and equally daunting downsides. Permanentdeletion of genes from the human genome is much more controversial thandeletion of bacterial genes. Thus, one desirable aspect of the presentinvention is directed to the far less controversial modification of gutmicrobes resident in the human being to promote health and to triggerthe desired immune responses as described herein.

CRISPR-Cas can be used on the various identified microbiome constituentsto modify gene expression, including cutting of a gene, repress oractivate a gene, etc. It can be employed to deliver desired regulatorsor any protein to a desired place on a genome of a microbe, thuspermitting one to tailor the attributes of the microbiome of anindividual to promote the health thereof, including the programmedtriggering of particular immune responses in an infant. BecauseCRISPR-Cas acts before transcription occurs, it is able to be employedto target regulatory and other elements on the DNA of microbes that makeup the microbiome. In certain embodiments, CRISPR-Cas is employed todeliver fluorescent markers to certain DNA sequences, thus permittingone to determine whether any particular sample has been treated inaccordance with the present invention, thus ensuring, for example,identity of various materials, safety issues, types of enhanced soils,etc. This permits labeling of living cells with a desired color. Stillother embodiments of the present invention are directed to the use ofstool samples from Amish to transplant to other humans, especiallyexpectant mothers, such that the attributes of the microbiome of theAmish can be enjoyed.

Certain embodiments rely upon the ability to deliver agents via mucosaladhesive strips, such as described, for example, in U.S. Pat. No.8,701,671, which is fully incorporated herein by this reference. Thus,in various embodiments of the present invention, the engineering ofcommunal bacteria with improved properties using a CRISPR/Cas system isemployed to provide for the enhancement of health. In certainembodiments the present invention is directed to delivering to microbialcells in vivo a delivery vehicle with at least one nucleic acid encodinga gene or nucleotide sequence of interest, such method employing anRNA-guided nuclease. The microbial cells may be either or bothpathogenic microbial cells or non-pathogenic bacterial cells and thegene or nucleotide sequence of interest may be a virulence factor gene,a toxin gene, an antibiotic resistance gene, or a modulatory gene, andmost preferably the nucleotide sequence of interest comprises 16Sribosomal DNA (rDNA). In certain embodiments, the delivery vehicle is abacteriophage. Thus, various embodiments of the present inventioninclude the use of CRISPR-Cas, with the recognition that this system canbe employed to benefit human health by modifying the bacterial and othermicrobe communities that humans have long been exposed to in a fashionsuch that the beneficial aspects of such microbes can be preserved,while the disadvantageous aspects can be “cut out” of the microbed\DNA—rather than attempting to change or modify the DNA of a human.

The present invention is one way in which the human health concerns canbe benefited directly by the use of a DNA deletion system withoutaffecting the long term and permanent deletion of human genes. It is notbelieved to be obvious, let alone intuitive, that human health can bebenefited by such a DNA deletion system used in a fashion that affectsonly gut microbes in a human's system. Moreover, the use of such a DNAmodification system for microbes, but not for the direct deletion ofgenes from a human, and the use of such a system prior to the exposureof a human to such modified microbes, has not previously been done,especially with the added step of modifying select microbes.

In still other embodiments, employment of technology described in U.S.Pat. No. 9,131,884 to Holmes, incorporated herein by this reference, isemployed to achieve desired further steps to address communication ofbiological disease status to a third party. For example, in certainembodiments, a medical device is associated with a mucosal strip thatcomprises a microarray having a bioactive agent capable of interactingwith a disease marker biological analyte and a reservoir having at leastone therapeutic agent, with the device able to release the therapeuticagent(s) from the medical device. In certain embodiments, at least twomicrochips with a microarray scanning device adapted to obtain physicalparameter data of an interaction between the disease marker biologicalanalyte and the bioactive agent is employed. A biometric recognitiondevice is configured to compare the physical parameter data with ananalyte interaction profile. The therapeutic agent releasing devicecontrols the release of the therapeutic agent from the reservoir. Theinterface device facilitates communications between the microarrayscanning device, biometric recognition device and the therapeutic agentreleasing device. An energy source to power the medical device can takeseveral forms, including biologically activated batteries that arepreferably associated with the strip.

In certain other embodiments, sugar is used as a source of energy,notably glucose that is converted into different sugars via an enzymaticcascade to provide necessary energy to create an electrochemicalgradient. This, in turn, is used to power an enzyme that synthesizesadenosine triphosphate (ATP). In contrast to natural catabolic pathwaysfor cellular glucose oxidation, a preferred embodiment does not rely onATP as an energy carrier. Instead, two redox enzymes oxidize glucose,generating reduced nicotinamide adenine dinucleotide (NADH) as the sugaris broken down. Another series of enzymes (as many as ten additionalenzymes) further breakdown the sugars and feed them back to the redoxenzymes to produce more NADH, with water and carbon dioxide being theonly by-products. NADH is a reducing agent and acts as an electronshuttle that carries electrons in living cells from one molecule toanother. NADH first transfers the electrons stripped from the glucose toa mediator with the help of an enzyme. The mediator then delivers theseelectrons to the battery's electrode, rendering it available to power anelectronic device. Such a battery mimics the way a living cell transferselectrons from one molecule to another to generate power, it runs onrenewable sugars, and has a high-energy storage density, rechargeableproviding an additional sugar solution. Malodextrin—a polymer made up ofglucose subunits—may be employed together with particular differentenzymes able to strip electrons from a single glucose molecule, thusharnessing the generated energy to power an electrical device. For suchembodiments, and to comply with written description and enablementrequirements, incorporated herein by the following references are thefollowing patent publications: 20150216917 to Jones; 20150361436 toHitchcock; 20150353901 to Liu; U.S. Pat. No. 9,131,884 to Holmes;20150064138 to Lu; 20150093473 to Barrangou; 20120027786 to Gupta;20150166641 to Goodman; 20150352023 to Berg.

Another aspect of the present invention includes the ability to load orimpregnate mucosal strips with any number of active agents to achieveother desirable aspects, such as administration of particular vitamins,medicinal components, and certain modified bacteria, thus treatingsymptoms as well as causes of GERD. For example, providing strips thatcontain compounds thereon that affect the ability of H. pylori to thrivein an individual's stomach is one aspect of various embodiments of thepresent invention. Other aspects involve the purposeful administrationof agents via an individual pressing with their tongue against an orallypositioned mucosal strip that has encapsulated packets of such agent.Thus, a new administration device is provided such that various aspectsof the present invention can be achieved by the use of such specializedstrips.

In still other embodiments, the use of additional ingredients mayprovide for chemical binding, and, for example, the use of liposometechnology can be employed. In some embodiments of the invention a partor all of the active ingredients on a mucosal adhesive strip areencapsulated within encapsulation structures selected to provide thedesired degree of adhesion to the mucous membranes of the throat, andadapted to release the active ingredients slowly over time in situ.These encapsulation structures may be distributed within the basematerial in the strip composition. In one embodiment, the encapsulationstructures comprise multilamellar microparticles. The multilamellarmicroparticles are selected to exhibit good adhesion to the mucousmembranes of the throat, and are small enough to be effectivelydistributed in the strip. The multiple layers may be structured to giveslow release of the active ingredient over the desired time period, sothat a single strip dose gives sustained activity over time, for exampleproviding for measurable activity for a sustained period of four or morehours, and ideally of for example 6 to 12 hours.

Preferably the strips of the present invention are made in a manner thatdo not dissolve in fewer than ten seconds, thus distinguishing the samefrom common breath strips widely available. The strips of the presentinvention may have a weight of from 30 to several hundred mg.,preferably over 33 mg. Preferably, strips of the present invention havesufficiently high moisture content to impart the product withflexibility and to avoid becoming brittle, e.g. the strips shouldpreferably avoid cracking when bent.

The strips of the present invention provide the requisite pliability andtensile strength necessary to securely adhere to a person's mucosaltissues for at least one hour, more preferably at least two hours, andeven preferably a bioadhesive polymer is selected from the groupconsisting of polycarbophil, carbomer, one or more acrylic polymers, oneor more polyacrylic acids, copolymers of these polymers, a water solublesalt of a co-polymer of methyl vinyl ether and maleic acid or anhydride,a combination thereof and their salts.

In certain embodiments, a mucosal adhesive strip has a coated surfacefor resisting bioadhesion that includes at least one patterned polymerincluding coating layer having a plurality of features attached to orprojected into a base surface. The features each have at least onemicroscale (<1 mm) dimension and have at least one neighboring featurehaving a substantially different geometry. The patterned coating layerpreferably provides an average roughness factor (R) of from 4 to 50. Thecoating layer resists or enhances bioadhesion as compared to the basesurface. An article having a surface coating with topography forcontrolling bioadhesion comprises a base surface, at least one patternedpolymer comprising coating layer including a plurality of spaced apartfeatures attached to or projected into the base surface which provide atleast a first feature spacing distance. The features each have at leastone microscale dimension and at least one neighboring feature having asubstantially different geometry. The coating layer provides an averageroughness factor (R) of from 2 to 50, preferably being from 4 to 50. Thecoating layer resists or enhances bioadhesion as compared to the basesurface.

Still other embodiments include the use of bacteria that have beenmodified to remove or disable one or more virulence factors of theparticular bacteria. In this regard, one aspect of the present inventionis directed to the modification of certain human-specific pathogens(including but not limited to H. pylori) by targeting one or morevirulence factors thereof, preferably by using CRISPR-Cas or CRISPR-Cpf1systems, to excise virulence factors genes, or at least portions thereofor transcriptional or translational controls therefore, such that suchpathogenic pathogens are deprived of their undesired pathogeniccharacteristics. One of skill in the art can readily assess the numberand identity of human-specific pathogens, as well as the particularvirulence factors associated therewith, and can then, employing theCRISPR systems as referenced herein, remove, render incapable orotherwise disable the virulence facts of such microorganisms such thatthey no long pose a pathogenic threat to humans. In various aspects ofthe present invention, there is a purposeful exposure of individuals tosuch modified pathogens such that the population of the same, forexample in the oral cavity or the human gut, competitively inhibits theinfection of non-modified pathogenic microbes of the same species. Otherembodiments provide for the delivery, via the strips as described hereinof one or more of the following microorganisms selected from the groupcomprising Lactobacillus lactis, Lactobacillus helveticus, Lactobacillusjensenii, Lactobacillus acidophilus, Lactobacillus bulgaricus,Lactobacillus amylovorus, Lactobacillus delbrueckii, Lactobacilluscasei, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillusjohnsonii, Lactobacillus paracasei, Lactobacillus pentosus,Lactobacillus rhamnosus, Lactobacillus curvatus, Lactobacillusplantarum, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillusfructivorans, Lactobacillus hilgardii, Lactobacillus fermentum,Lactobacillus reuteri, Lactobacillus viridescens, Bifidobacteriumbifidum, and Lactobacillus ingluviei.

Moreover, in preferred embodiments, the microbes modified are limited tothose demonstrating human tropism such that undesired and unintendedchanges to other animals and organisms are not affected and that theonly implications of such genomic alterations of human specificpathogens are restricted to such species in a manner that is not capableof affecting other than the particular human disease at issue. This caninclude, for example, modifications and/or employment of integrons,which are a two-component genetic recombination system present in thechromosome of many bacterial species. The integron incorporates mobilegenes termed gene cassettes into a reserved genetic site viasite-specific recombination, named the Integron/gene cassette system.The integron consists of three basic elements: an integrase gene, anattachment site and a promoter. These elements can be manipulated to,for example, decrease the ability of a particular bacteria in a person'sgut from being able to effectively attach to epithelial tissue; oralternatively, to coaggregate with other bacteria.

To provide necessary and sufficient written disclosure and enablement ofthe various embodiments of the present invention, the followingreferences are incorporated by reference in their entireties: U.S. Pat.No. 9,017,718 to Tan; 20140065218 to Lang et. al.; U.S. Pat. Nos.6,599,883; 8,383,201; 5,158,789; 20070218114 to Sorousch; 20040136923 toDavidson; U.S. Pat. No. 8,999,372 to Davidson; 20090196907 to Bunick;20090196908 to Lee; 20030124178 to Haley; 20070293587 to Haley;20100285098 to Haley; 2006-0204591 to Burrell; U.S. Pat. No. 7,087,249to Burrelll; U.S. Pat. No. 6,210,699 to Acharya; U.S. Pat. No. 8,865,211to Tzannis; 20140199266 to Park; U.S. Pat. No. 6,599,883 to Romeo;PCT/US2008/080362 to Dussia; 2007-0218114 to Duggan; 2004-0136923 toDavidson; 20110142942 to Schobel; 20040120991 to Gardner et al.; Fuchset al. U.S. Pat. No. 4,136,162; 20040136923 to Davidson; U.S. Pat. No.4,163,777 to Mitra; U.S. Pat. No. 5,002,970 to Eby, III; 20040096569 toBarkalow et al.; 20060035008 to Virgallito et al.; 20030031737 toRosenbloom; U.S. Pat. No. 6,919,373 to Lam et al.; 20050196358 toGeorglades et al.; U.S. Pat. No. 3,832,460 to Kosti; 2002002057 toBattey et al.; 20040228804 to Jones, et al.; U.S. Pat. No. 6,054,143 toJones; U.S. Pat. No. 5,719,196 to Uhari; 20150150792 to Klingman;20140333003 to Allen; 20140271867 to Myers; 20140356460 to Lutin;20150038594 to Borges; U.S. Pat. No. 6,139,861 to Friedman; 20160206564to Trachtman; 20160089405 to Lue; and U.S. Pat. No. 7,901,925 to Bojrab.

Another aspect of the certain embodiments of the present invention isdirected to a thin film mucosal layered strip that includes severallayers, and in one particular embodiment, at least four layers, with afirst layer comprising an odor impervious material, a second layer thatcomprises at least one encapsulated solvent, a third layer having asolvent absorbent material, and a fourth layer comprising an adhesive,wherein the solvent is encapsulated in a frangible enclosure and ispresent in an amount of at least about 0.5 ml.

Treatment agents can be encapsulated in such strips, such thatantibiotics or co aggregation agents or LAB, etc. can be encapsulated ina manner that they can be released into the oral cavity at a time whenthe person so desires and/or when the strip dissolves to a certainextent, e.g. when the walls of the encapsulating shell is worn thinenough to fracture to release the agent(s). The manner in which acapsule can be fractured in order to release its solvent contents isvariable and will be understood by those of skill in the art.Preferably, the capsule is constructed in a manner that it issufficiently robust such that mere transport and packaging of the stripscontaining such capsules does not cause any leakage or breakage of suchcapsules. Instead, the design of capsules is such that they arefrangible with a considerable amount of force being directly appliedthereto once the strips are placed on a particular mucosal surface, suchas on the soft palette of a human, such that the person's tongue, whenpressing against such capsule, can cause it to fracture to release thecontents of the capsule. In other embodiments, two or more differentmaterials may be released.

The CRISPR-Cas system is preferably employed to excise the virulencefactors of one or more of the following bacteria: Lactobacillus lactis,Lactobacillus helveticus, Lactobacillus jensenii, Lactobacillusacidophilus, Lactobacillus bulgaricus, Lactobacillus amylovorus,Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus crispatus,Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei,Lactobacillus pentosus, Lactobacillus rhamnosus, Lactobacillus curvatus,Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri,Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillusfermen turn, Lactobacillus reuteri, Lactobacillus viridescens,Bifidobacterium bifidum, Lactobacillus ingluviei and preferably selectedfrom the group comprising the following microorganisms deposited withthe German Collection for Microorganisms and Cell Cultures where theyare numbered as DSM 25972, DSM 25987, DSM 25988, DSM 25989, DSM 25973and have been in accordance with the Budapest Treaty regardingInternational Recognition of the Deposition of Microorganisms for thepurpose of patent deposition. In a preferred embodiment of theinvention, strips containing effective amounts of these bacteria areprovided that are attached to the soft palate region of a person's mouthor on other mucosal surfaces. Other LAB that may be employed in variousembodiments include the following: lactobacillus slaivarius CICC 23174;lactobacillus plantarum CGMCC 1.557, lactobacillus rhamnosus ATCC 53103,and lactobacillus acidophilus ATCC 4356.

The timeliness of being able to address an imminent cellular interactionby providing medications or beneficial agents directly to the tissue atissue by merely having one's tongue cause a frangible vesicle,encapsulation, etc. to release a desired agent to contact with thetissue, is a novel way in which to effectively stop a biological processbefore it progresses to a later stage, at which point the desired effectof the agent may not be efficacious.

Yet another embodiment of the present invention is directed to themodification of certain bacteria such that they achieve the desiredproduction of acid in an individual's stomach, but without causing GERD.Thus, providing modified bacteria that produce gastrin, which triggersparietal cells in the stomach lining to produce more hydrochloric acid,is achieved so that invading microbes are killed. While H. pylori can bemodified to achieve this feat, other bacteria can be modified to survivein the acid environment of the stomach, while also avoiding thedetrimental aspects of native H. pylori. The objective is to populatethe stomach microbiome with microbes that thrive in the gastric acidenvironment but without interfering with the stomach's natural defensemechanism. An abnormally low level of acidity in the stomach is a factorin various disease states.

It is believed that microorganisms residing in the GI tract can createchemicals that affect portions of the nervous system and muscle tissuesin the GI tract that control the sphincter valve at the entrance of thestomach from the esophagus. Thus, certain embodiments of the presentinvention are directed to the purposeful administration to anindividual's GI tract of modified bacteria, e.g. via CRISPR systems, togenerate desired chemicals that control the sphincter valve in a mannerto address GERD. Thus, in accordance with one or more embodiments, aneffective amount of a medicament comprised of one or more suitablebacteria is administered to a person to prevent, mitigate or treat GERD,such bacteria comprising, e.g. one or more Lactobacillus, Prevotella, H.pylori, etc. Moreover, in certain embodiments, in addition to modifiedbacteria in an oral strip, such strip may also include desired amountsof drugs, such as PPI's. Thus, thin oral solvent cast films for thedelivery of the proton pump inhibitor, omeprazole (OME) via the buccalmucosa can be provided.

One of skill in the art will appreciate that still other embodiments ofthe present invention are directed to aspects of human health that aresimilarly related to the proper balance and populations of bacteria inan individual's microbiome, and in particular, to the reduction in thelikelihood of developing, preventing and treating of Parkinson'sdisease. Parkinson's disease is a neurodegenerative disordercharacterized by a chronic and progressive loss of dopamine neurons insubstantia nigra pars compacta, leading to movement disorders includingdyskinesia, resting tremor, rigidity, and gait disturbance. Parkinson'sdisease is the second most common neurodegenerative disease worldwideand affects more than one percent of people older than sixty years ofage and roughly four percent of those older than 85. Although causalityof the disease is not known, chronic inflammation appears to play animportant role in the pathogenesis of Parkinson's disease. Significantincrease of inflammatory cytokines such as TNF-alpha, Il-1beta andIFN-gamma in glial cells in the substantia nigra of Parkinson's patientshas been observed. One aspect of the present invention is directed toreducing the likelihood of developing Parkinson's disease by modulationof an individual's microbiome. Reduction of certain bacteria in aperson's microbiome correlates to a reduction in the likelihood that onewill develop Parkinson's disease. Such bacteria include Proteus sp.,Bilophila sp., and Roseburia sp. To avoid Parkinson's disease,increasing the populations of members of the microbiological familiesLachnospiraceae, Rikenellaceae, and Peptostreptococcaceae, andButyricicoccus sp. is a suggested way to prevent or treat the disease.Thus, in several embodiments, the present invention is directed togene-microbiome interactions that address the etiology for Parkinson'sdisease by targeting the gut bacteria to promote enhanced motor control.Distinct microbes associated with Parkinson's disease, rather thangeneral microbial stimulation, manifest disease symptoms. Increasing thepopulations in an individual suffering from Parkinson's disease from thebacteria family Lachnospiraceae and Ruminococceae and lowering theamount of gut microbiomes of Proteobacteria is one aspect of particularembodiments of the invention. With one goal being to achieve elevatedrelative concentration of butyrate in the gut. Thus, in general atreatment for Parkinson's disease involves the provision to anindividual's microbiota bacteria that are found to be missing or inreduced numbers as compared to healthy individuals, and the reduction ofpathogenic resident microbes in such patients. Parkinson's disease isbut one of several diseases that involve the gut bacteria as a link todisorders, e.g. such as anxiety, depression, and autism, thus, variousneurologic conditions are able to be treated by using the invention asset forth herein to address such conditions. For example, in variousembodiments, oral administration of specific microbial metabolites toaffect the ability of gut bacteria to regulate movement disorders is oneobjective of the present invention.

In still other particular embodiments, as discussed above with respectto the connections between the oral and gut microbiomes and the variousdiseases stemming from dysbiosis thereof, by controlling theinflammatory response associated with gingivitis, the subgingivalnutrient supply to the subgingival microbiota will not be conducive toperiodontal pathogen overgrowth. Thus, by addressing the inflammatoryresponse of an individual, it is possible to control infection bymodulating the subgingival microbiota of an individual so that it ispredominated by commensal bacteria compatible with periodontal health.By maintaining periodontal health, the occasions for pathogenic bacteriato travel to other parts of the body, including the brain and bloodvessels, one is able to prevent various diseases, including but notlimited to Parkinson's disease, Alzheimer's Disease, heart disease,GERD, etc.

Other aspects of the present invention relate to the modulation of anindividual's microbiome by the use of dietary supplements, for example,by the use of omega-3 polyunsaturated fatty acids, well recognized fortheir anti-inflammatory properties, in combination with the provision ofbeneficial bacteria as described herein. Still other embodiments aredirected to the modulation of an individual's microbiome by theadministration of particular antibiotics (often followed by thepurposeful administration of bacteria to one's microbiome), such as asystemic antibiotic (tetracycline) and especially in combination with anonsteroidal anti-inflammatory drug (e.g. ibuprofen). Such combinationsare often administered in connection with the treatment of periodontaltreatments, such as with non-surgical root surface debridement to reducecertain oral microbiome communities and to alter the same in a way topromote health. Other agents can be employed in various embodiments ofthe present invention, including the use of azithromycin, whichpossesses antibacterial and also exerts anti-inflammation properties,thus allowing a combination of antibiotic and anti-inflammatorycapabilities with one agent.

While specific embodiments and applications of the present inventionhave been described, it is to be understood that the invention is notlimited to the precise configuration and components disclosed herein.Various modifications, changes, and variations which will be apparent tothose skilled in the art may be made in the arrangement, operation, anddetails of the methods and systems of the present invention disclosedherein without departing from the spirit and scope of the invention.Those skilled in the art will appreciate that the conception upon whichthis disclosure is based, may readily be utilized as a basis fordesigning of other methods and systems for carrying out the severalpurposes of the present invention to instruct and encourage theprevention and treatment of various human diseases. It is important,therefore, that the claims be regarded as including any such equivalentconstruction insofar as they do not depart from the spirit and scope ofthe present invention.

What is claimed is:
 1. A method for reducing the likelihood of GERD inan individual, comprising: administering to an individual an amount ofH. pylori modified by a CRISPR-Cas or Cpf1 system to render H. pylorimore susceptible to antibiotics; permitting said modified H. pylori tobecome the dominant population of H. Pylori in the individual's stomach;and administering an effective amount of an antibiotic effective to killthe modified H.
 2. The method as set forth in claim 1, furthercomprising affecting the ability of H. pylori to form biofilms bymodifying its ability to produce lipid A.
 3. The method as set forth inclaim 1, wherein the CRISPR-Cas or Cpf1 system is used to interfere withgenes involved in the formation of biofilms by H. pylori.
 4. The methodas set forth in claim 1, wherein the CRISPR-Cas or Cpf1 system isemployed to modify only bacterial genes of bacteria that reside in thehuman gut that are non-homologous to those encompassed in the humangenome.
 5. The method as set forth in claim 1, further comprising usingthe CRISPR-Cas or Cpf1 system to excise particular characteristics of C.difficile to reduce the release of phage particles.